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On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies

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Abstract

NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and 13C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.

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Acknowledgements

This project was supported by the National Science Foundation (Grant 1000136529 to L.D.C.), the American Heart Association (Grant 16PRE27200004 to L.D.C.), the Welch Foundation (I-1770 to D.M.R), a Packard Foundation Fellowship (to D.M.R.), the Mallinckrodt Foundation (to D.M.R), and the National Institutes of Health (F32 GM119311 to I.D., R01 GM113050 to D.M.R., and R01 GM106239 to K.H.G.). Reagents for NMR spectroscopy were generously provided by a research award from Cambridge Isotope Laboratories, Inc.

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Authors and Affiliations

  1. Department of Biophysics, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX, 75390-8816, USA

    Lindsay Clark & Daniel M. Rosenbaum

  2. Molecular Biophysics Graduate Program, The University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA

    Lindsay Clark & Daniel M. Rosenbaum

  3. Structural Biology Initiative, CUNY Advanced Science Research Center, 85 St. Nicholas Terrace, New York, NY, 10031, USA

    Igor Dikiy & Kevin H. Gardner

  4. Department of Chemistry and Biochemistry, City College of New York, New York, NY, 10031, USA

    Kevin H. Gardner

  5. Biochemistry, Chemistry and Biology Ph.D. Programs, Graduate Center, City University of New York, New York, NY, 10016, USA

    Kevin H. Gardner

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  1. Lindsay Clark
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  2. Igor Dikiy
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  3. Daniel M. Rosenbaum
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Correspondence to Daniel M. Rosenbaum or Kevin H. Gardner.

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Clark, L., Dikiy, I., Rosenbaum, D.M. et al. On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies. J Biomol NMR 71, 203–211 (2018). https://doi.org/10.1007/s10858-018-0204-3

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